Prostacyclin Enhances Stretch-induced Surfactant ... - ATS Journals

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FRANK ROSE, KLAUS ZWICK, HOSSEIN ARDESCHIR GHOFRANI, ULF SIBELIUS, WERNER SEEGER,. DIETER WALMRATH, and FRIEDRICH GRIMMINGER.
Prostacyclin Enhances Stretch-induced Surfactant Secretion in Alveolar Epithelial Type II Cells FRANK ROSE, KLAUS ZWICK, HOSSEIN ARDESCHIR GHOFRANI, ULF SIBELIUS, WERNER SEEGER, DIETER WALMRATH, and FRIEDRICH GRIMMINGER Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany

Inhalative vasodilator therapy, employing gaseous nitric oxide (NO) or aerosolized prostaglandin PGI2, is of interest for regional pulmonary vasodilation in ARDS and pulmonary hypertension. We investigated the impact of the NO donor spermine NONOate as well as PGI2 and its stable chemical analog iloprost on cultured rat alveolar epithelial type II cell (ATII) surfactant secretion. The NO donor provoked a significant increase in the ATII cGMP content, further enhanced by type V phosphodiesterase (PDE) inhibition, but affected neither baseline nor mechanical stretch-induced surfactant secretion. The prostanoids caused a marked increase in the epithelial cAMP content, further amplified by coadministration of type III/IV PDE inhibitors. Baseline surfactant secretion was not altered by this approach, but mechanical stretch-induced liberation of surfactant was significantly increased, most prominently in the ATII with the highest cAMP levels due to the presence of both iloprost and PDE III/IV inhibitors. In contrast, epithelial phosphoinositide metabolism, well responsive to purinergic stimulation as positive control, was unchanged in prostanoid-exposed cells. We conclude that the PGI2–cAMP axis, but not the NO–cGMP axis, forwards a markedly enhanced secretory response to the physiological stimulus of cell surface stretching, which may be relevant for therapeutic use of these agents. Rose F, Zwick K, Ghofrani HA, Sibelius U, Seeger W, Walmrath D, Grimminger F. Prostacyclin enhances stretch-induced surfactant secretion in alveolar epithelial type II cells. AM J RESPIR CRIT CARE MED 1999;160:846–851.

Deep lung inflation to total lung capacity provokes substantial stretching of the alveolar epithelial barrier, with up to 80% area increase, and is regarded as the predominant physiological stimulus for surfactant secretion in alveolar epithelial type II cells (ATII; type II pneumocytes), a process that is fundamental for maintaining alveolar stability and lung gas exchange (1). At the interface between external environments and the interior milieu, the alveolar lining layer, this cell type releases both lipid and protein surfactant components from cytoplasmic organelles called lamellar bodies (1–3). Alterations of the alveolar surfactant system have long since been implicated in the pathogenic sequelae of severe pneumonia, acute respiratory distress syndrome, and chronic interstitial lung diseases. Lack of essential surfactant compounds, in particular the tubular myelin fraction and the hydrophobic surfactant apoproteins, was suggested to contribute to these alterations and might reflect changes in the secretion profiles of ATII cells under these conditions (4, 5). Inhalation of nitric oxide (NO) and nebulization of pros-

(Received in original form December 29, 1998 and in revised form March 22, 1999) Supported by the Deutsche Forschungsgemeinschaft (SFB 547 Cardiopulmonary Vascular System). Correspondence and requests for reprints should be addressed to F. Grimminger, M.D., Ph.D., Department of Internal Medicine, Klinikstraße 36, D-35392 Giessen, Germany. Am J Respir Crit Care Med Vol 160. pp 846–851, 1999 Internet address: www.atsjournals.org

taglandin I2 (PGI2) or its long-acting analog iloprost are new strategies for selective pulmonary vasodilation in acute respiratory failure and pulmonary hypertension (6–12). Such treatment targets regional vasodilation in well-ventilated lung areas, thereby reducing shunt flow and improving arterial oxygenation. Beyond doubt, however, this approach of vasodilator administration will provoke high concentrations of these agents at the interface itself, including the alveolar lining layer and the epithelial cells. Concerning the extracellular components of the surfactant system, high concentrations of NO were noted to deteriorate biophysical surfactant function, which might be ascribed to NO-related peroxynitrite formation (13– 17). Moreover, NO was found to decrease ATP content and surfactant synthesis in freshly isolated ATII cells (18, 19). The present study investigates the impact of NO versus prostanoids on the surfactant secretory mechanisms in type II pneumocytes. Doses of NO donors and PGI2 or iloprost were chosen to mimic concentrations of the vasodilators assumed to arise in the alveolar lining layer during inhalative therapy. Against the background of NO signal transduction via cGMP and prostanoid signal transduction via cAMP (20, 21), the appearance of these second messengers was addressed and phosphodiesterase inhibitors were, in addition, employed for stabilization of these cyclic nucleotides (22, 23). Moreover, ATII phosphoinositide metabolism, also implicated in surfactant secretory mechanisms (2), was analyzed in the presence of these agents. In essence, prostanoid treatment of ATII cells resulted in a marked increase in the stretch-induced surfactant secretion, coincident with enhanced cAMP accumulation, but

Rose, Zwick, Ghofrani, et al.: Stretch, Vasodilators, Surfactant Secretion in Pneumocytes

not phosphoinositide breakdown. Both cAMP accumulation and surfactant secretion were further increased on coapplication of type III and IV phosphodiesterase inhibitors. In contrast, nitric oxide donors failed to modulate the stretch-induced surfactant release. These data suggest that NO and nebulized PGI2, both employed for inhalative vasodilator therapy, exert differential impacts on the surfactant secretory mechanisms of ATII cells.

METHODS Male CD18 Sprague-Dawley rats (180–200 g) were purchased from Charles River (Sulzfeld am Main, Germany). Elastase (type EC 134; specific activity, 135 U/mg protein) was purchased from Elastin Products Company (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) was supplied by GIBCO (Karlsruhe, Germany). Isobutyl methylxanthine (IBMX), rolipram, and zaprinast were obtained from Sigma (Deisenhofen, Germany). Zardaverine was kindly provided by C. Schudt (Byk Gulden GmbH, Konstanz, Germany), iloprost was obtained from Schering (Berlin, Germany), and Flolan (PGI2) was from Glaxo-Wellcome (Hamburg, Germany). S-Nitroso-N-acetyl-DLpenicillamine and spermine NONOate were purchased from Calbiochem-Behring (La Jolla, CA). [3H]Methylcholine, myo-[2-3H]inositol, and the 125I-labeled cAMP and 125I-labeled cGMP assay systems were from Amersham (Dreieich, Germany). Tissue culture plastic was purchased from Becton Dickinson (Heidelberg, Germany). Bio Flex culture plates (35-mm-diameter silicone membranes, six well) were obtained from Flexcell International Corporation (Hillsborough, NC).

Isolation of Type II Alveolar Epithelial Cells ATII cells were isolated as previously described in detail (1). Briefly, inflated and perfused lungs from specific pathogen-free male CD18 Sprague-Dawley rats were lavaged and filled to their total lung capacity with solution containing elastase (30 U/ml) and trypsin (0.05 mg/ ml). Lungs were minced and free cells were separated from lung tissue by sequential filtration through sterile gauze (100-mm poresize), and additional 10-mm pore size nylon mesh. “Panning” of the resultant cell suspension was performed on rat IgG-coated plates. Nonadherent ATII cells were harvested after 1 h and resuspended in DMEM containing 10% fetal calf serum (FCS). The yield of type II epithelial cells from each rat was in the range of 30–50 3 106. The percentage of type II cells was 94 6 2% as assessed by modified Papanicolaou, tannic acid, and alkaline phosphatase staining. Contaminated cells included alveolar macrophages (less than 4% in all experiments), and neutrophils (less than 2%). ATII cell viability, as assessed by 5-carboxyfluorescein diacetate (CFDA) loading and trypan blue exclusion, was persistently greater than 95%. Lactate dehydrogenase (LDH) release as one indicator of cellular damage was less than 2% of total enzyme activity as compared with total release in response to the pore-forming agent mellitin (100 g/ml). No significant change was noted in LDH release by cells from unstretched or stretched control membranes.

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lated cells were seeded on silicone membranes (six-well Bio Flex culture plates) coated with fibronectin. Culture plates were placed on a loading post and vacuum was applied to the bottom of the plates, symmetrically increasing the surface of the cells. Membranes were maintained in the distended state for about 30 s. The amount of cell distention (defined as the extent of the cell in two dimensions) was determined by microscopic photography, with adjacent measurement of the corresponding cell diameter during projection on a screen. A mean increase in the cell surface area to 114–115% was chosen. In each experiment, duplicate paired plates were either subjected to stretch or maintained unstretched as a control. Separate control experiments involving thin-layer chromatography ascertained that the radioactive lipids secreted from both unstimulated and stimulated cells consisted of 93 6 3% phosphatidylcholine (data not given in detail).

Measurement of cAMP/cGMP ATII cells were cultured on 35-mm dishes at a density of 3 3 106 cells per well (plating density was typically 4 3 105/cm2). Measurement of cyclic nucleotides was performed using commercially available 125Ilabeled cAMP and 125I-labeled cGMP radioimmunoassays to analyze the supernatant of permeated cells subjected to 70% ethanol for 60 min.

Phosphoinositide Metabolism The phosphatidylinositol (PtdIns) turnover of stimulated ATII cells was investigated by measuring the accumulation of inositol phosphates according to Berridge and coworkers (25). Cells were cultured on 35-mm dishes at a density of 3 3 106 cells per well. Cellular phospholipid pools were fed with myo-[3H]inositol (10 mCi/well) in DMEM containing 2% fetal calf serum plus 40 mM HEPES buffer (pH 7.4) and incubated at 378 C for 12 h. Before experimental use, cells were washed twice in Hanks’ balanced salt solution containing 20 mM HEPES and 10 mM LiCl. At various times after stimulus application, samples were quenched with trichloracetic acid (final concentration, 7.5%), kept on ice for 15 min, and extracted four times with diethyl

Determination of Surfactant Secretion Freshly isolated cells were seeded at densities of 5 3 105 and 1 3 106 cells per well on 12- and 6-well culture dishes, respectively. The plating density was typically 8 3 104/cm2 (z 64% plating efficiency). After prelabeling with [3H]choline (1 mCi/ml) for 18 h, the cells were washed and medium was replaced with unlabeled serum-free DMEM, containing agents under investigation. After incubation for preset time periods, medium was removed and centrifuged to sediment possibly detached cells, which were not retrieved and used in the assays. The monolayer was scraped off and lipids from both medium and cells were extracted according to the Folch partition (24). The amount of phosphatidylcholine (PC) secretion was calculated as the percentage of [3H]PC contained in the medium as related to the total amount of [3H]PC in cells plus medium.

Mechanical Stretch of Type II Alveolar Epithelial Cells To determine the effect of mechanical distention on surfactant secretion by ATII cells, we used an equibiaxial stretching device (Bioflex strain unit system; Flexcell International Corporation). Freshly iso-

Figure 1. Impact of iloprost pretreatment on stretch-induced [3H]PC secretion. Pretreatment with different concentrations of iloprost or sham pretreatment was performed for 15 min. Next, one 30-s stretch was performed in one-half of the experiments, and the [3H]PC secretion was assessed after 1 h. With reference to secretagogue-induced [3H]PC secretion, ATII cells were either incubated with ATP (5 mM) or terbutaline (100 mM), or sham incubated for 1 h (basal). Data on [ 3H]PC secretion are given as the percentage of secreted tracer in relation to the total amount of tracer contained in cells plus medium. #Significantly different from stretched cells in the absence of iloprost. Means 6 SEM of six independent experiments, each with separate ATII preparations, are presented.

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ether. The aqueous phase was neutralized with sodium tetraborate to pH 8.0, and processed to separate inositol phosphates on Dowex anion-exchange columns as described by Berridge and colleagues (25). The column was eluted sequentially with water (for free [3H]inositol), 5 mM sodium tetraborate–60 mM sodium formate (for glycerophospho-[3H]inositol), 0.1 M formic acid–0.2 M ammonium formate (for [3H]inositol monophosphate; [3H]IP1), 0.1 M formic acid–0.5 M ammonium formate (for [3H]IP2), and 0.1 M formic acid/1.0 M ammonium formate (for [3H]IP3); and samples were processed for liquid scintillation counting.

Statistical Analysis For statistical comparison, one-way analysis of variance was performed. A level of p , 0.05 was considered significant.

RESULTS Influence of Prostacyclin on Surfactant Secretion in ATII Cells

Exposure of ATII cells to stretching in an equibiaxial stretching device caused a nearly 50% increase in surfactant secretion, as compared with baseline (Figure 1). This secretory response was comparable to that evoked by 5 mM ATP, and even surpassed that elicited by 100 mM terbutaline. Increasing concentrations of the stable PGI2 analog iloprost did not influence the baseline surfactant secretion by ATII cells assessed under nonstretched conditions. In contrast, when ATII cells were stretched in the presence of increasing doses of iloprost, a marked enhancement of surfactant secretion was noted. At the maximum concentration of iloprost employed (250 ng/ml), the [3H]dipalmitoylphosphatidylcholine ([3H]DPPC) release was about 1.5 times higher then the control values assessed in stretched cells in the absence of prostanoid.

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of inositol phosphates (Figure 2). The prostanoid did, however, provoke a dose-dependent increase in the ATII cAMP content (Figure 3). This metabolic response approached z 50% of that elicited by the potent adenylate cyclase activator forskolin. Maximum cAMP levels were detected within 30 min after incubation with iloprost, with subsequent decline to near baseline levels within 60 min. Preceding repetitive iloprost administration only slightly increased and prolonged the cAMP response to the prostanoid. ATII incubation with the cAMP-specific phosphodiesterase (PDE) inhibitors rolipram (specific for PDE type IV) and zardaverine (specific for PDE types III and IV) provoked a dose-dependent augmentation of the intracellular cAMP content within 15 min to three- to fourfold increased values above baseline (data not given in detail). Most prominent enhancement of the cAMP response was also noted on coapplication of iloprost with low concentrations of rolipram (Figure 3). Under these conditions, no decline in the ATII cAMP levels within 60 min was noted. Influence of Nitric Oxide and Phosphodiesterase Inhibitors on the Intracellular cGMP Content in ATII Cells

Incubation of ATII cells with the exogenous NO donor spermine NONOate caused a dose-dependent increase in the ATII cGMP level (Figure 4). This response was amplified in the presence of the specific PDE type V inhibitor zaprinast and the unspecific inhibitor IBMX. Phosphodiesterase Inhibition, Iloprost- and Nitric Oxide–induced Surfactant Secretion

When given as sole agent, low concentrations of rolipram (0.1 mM, 15 min) did not increase the ATII surfactant secretion.

Influence of Prostacyclin on cAMP Content and Inositol Phosphates in ATII Cells

In contrast to ATP employed as a positive control, iloprost failed to cause phosphoinositide hydrolysis with accumulation

Figure 2. Time course of inositol phosphate generation in response to ATP and iloprost. ATII cells, prelabeled with [3H]inositol, were incubated with ATP (50 mM) and iloprost (10 and 100 ng/ ml) for various time periods. Extracted inositol phosphates were separated by anion-exchange chromatography. IP 3, IP2, and IP1 are collectively depicted as IPx. #Significantly different from control. Means 6 SEM of four independent experiments, each with separate ATII preparations, are presented.

Figure 3. Time course of cAMP generation in response to iloprost. ATII cells were stimulated repetitively with iloprost (100 ng/ml) every 15 min or once with iloprost at time zero, or sham incubation was performed. In one part of the experiments pretreatment with low-dose rolipram (0.1 mM) or sham pretreatment was performed for 15 min. Next, ATII cells were incubated with iloprost (100 ng/ ml) and forskolin (2.5 mM) for 15 min or sham incubated. cAMP release into the supernatant of permeated cells was quantified using a commercially available 125I-labeled cAMP radioimmunoassay. # Significantly different from control. Means 6 SEM of four independent experiments, each with separate ATII preparations, are presented.

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Figure 4. Impact of zaprinast and IBMX pretreatment on agonistinduced cGMP generation. Pretreatment with zaprinast (10 mM) and IBMX (60 mM) or sham pretreatment was performed for 15 min. Next, ATII cells were incubated without or with various concentrations of NONOate for 30 min. cGMP release into the supernatant of permeated cells was quantified using a commercially available 125I-labeled cGMP radioimmunoassay. Means 6 SEM of five independent experiments, each with separate ATII preparations, are presented.

Pretreatment with the PDE blocker, however, significantly enhanced the forskolin (5 mM)-evoked secretory response within 2 h from 1.34 6 0.14 to 1.9 6 0.2% PC secretion (means 6 SEM of four independent experiments each). Such marked enhancement of surfactant secretion was also noted on coapplication of iloprost and low-dose rolipram in stretched ATII cells (Figure 5). Corresponding results were obtained when iloprost was replaced by the short-acting PGI2, coadministered with the PDE III/IV inhibitor zardaverine (Table 1). In contrast, even high concentrations of NONOate did not influence baseline and stretch-induced ATII surfactant secretion, either in the absence or presence of zaprinast. Control Experiments

To measure the mechanical distention, the membranes remained in the equibiaxial distended state for about 30 s. The routine use of a cell surface area increase to 115% did not provoke obvious type II cell damage, as assessed by microscopy performed periodically to determine cell adhesion and membrane integrity, as well as by measurement of LDH as a marker of overt cell lysis: the currently employed mode of cell stretching did not evoke significant LDH release (0.5–1% of total cellular LDH was liberated within 60 min both in nonstretched controls and in ATII cells exposed to the stretch procedure). In addition, the stretch-induced increase in surfactant release was reduced to near baseline values in the presence of 1 mM surfactant protein A (SpA), indicating that the cell integrity was not destroyed mechanically (data not shown).

DISCUSSION The present study, using an equibiaxial stretching device, corroborates previous ATII cell data collected by Wirtz and Dobbs (1), in showing that a single stretch maneuver causes an approximately 50% increase in surfactant secretion, a response that is of fundamental physiological significance. The underly-

Figure 5. Impact of either iloprost and rolipram or NONOate and zaprinast pretreatment on stretch-induced [ 3H]PC secretion. Pretreatment with different concentrations of iloprost, rolipram (0.1 mM), NONOate, or zaprinast (10 mM) or sham pretreatment was performed for 15 min. Next, one 30-s stretch was performed in parts of the studies, and the [ 3H]PC secretion was assessed after 1 h. Data on [3H]PC secretion are given as the percentage of secreted tracer in relation to the total amount of tracer contained in cells plus medium. #Significantly different from stretched and iloprost-incubated cells in the absence of rolipram. Means 6 SEM of six (iloprost and rolipram) and four (NONOate and zaprinast) independent experiments, each with separate ATII preparations, are presented.

ing signaling events are, however, not yet fully disclosed, in particular the early steps of mechanotransduction finally resulting in the rapid extrusion of lamellar bodies. The current hypothesis, as depicted in Figure 6, centers around a stretchinduced transient increase in cytoplasmic calcium that may be linked to phosphoinositide metabolic steps (1, 2, 26, 27). Receptor binding of prostacyclin is coupled to signaling events in many cell types, with cAMP formation and accumulation of inositol phosphates representing the predominant second-messenger pathways (21). The stimulation of the adenylate cyclase pathway is predominantly linked to the occupancy of the IP receptor type, with coupling proceeding via G-stimulatory protein (Gs) (21, 28). Binding of PGI2 to EP1 receptors, in contrast, is considered to be predominantly linked to phosphoinositide hydrolysis-related signal transduction. The PGI2 analog iloprost combines chemical stability with high IP and EP1 receptor agonist potency (21, 28). IP receptor message was found to be abundant in lung tissue (29). It is well in this line that iloprost markedly increased the ATII cAMP

TABLE 1 IMPACT OF PGI2 AND ZARDAVERINE PRETREATMENT ON STRETCH-INDUCED [3H]PHOSPHATIDYLCHOLINE SECRETION*

Nonstretched Stretched

Control

Zardaverine

PGI2

Zardaverine 1 PGI2

100 210 6 21

105 6 24 215 6 26

105 6 12 290 6 26

145 6 13 395 6 46

* Pretreatment with PGI2 (400 mM) or zardaverine (0.1 mM), or sham pretreatment, was performed for 15 min. Next, one 30-s stretch was undertaken in part of the studies, and the [3H]PC secretion was assessed after 1 h. All data on [3H]PC secretion, under stretched and nonstretched conditions, are given as percentages of the baseline in nonstretched controls (set 100%).

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Figure 6. Schematic representing the postulated signal transduction pathways investigated in this study. Both iloprost-induced cAMP formation and calcium release cooperate in the surfactant secretory response. Stabilization of the intracellular cAMP level is achieved by cAMP-specific phosphodiesterase inhibitors. In contrast, the NO–cGMP signaling pathway does not enhance (but might rather inhibit) surfactant secretion.

content in a dose-dependent manner, with peaking of the cyclic nucleotide level after z 15–30 min and subsequent rapid decline to near baseline values within 1 h. In contrast, no effect of iloprost (and PGI2, data not given) on the ATII phosphatidylinositol hydrolysis signaling pathway was noted, as the inositol phosphate levels remained fully unchanged. According to the current understanding of prostanoid receptor-binding events (21, 28), this observation suggests predominant if not exclusive employment of the IP receptor pathway in type II pneumocytes responding to PGI2 and iloprost. We preferred iloprost over PGI2 itself to overcome the transient feature of ATII stimulation in response to the chemically unstable native prostanoid. However, in spite of the chemical stability of iloprost, known to underlay a much longer half-life in vivo as compared with PGI2 (10, 21), rapid decline of the pneumocyte cAMP content within 60 min was noted. Moreover, even additional repetitive iloprost administrations during this time period did not overcome this desensitization phenomenon. This observation is reminiscent of the desensitization of platelets to prolonged iloprost incubation, which was shown to be due to IP receptor internalization with corresponding downregulation of prostanoid responsiveness (30, 31). Interestingly, the internalized receptors appear not to be degraded but may be rapidly recycled to the platelet surface in functionally active form after withdrawal of the prostanoid, a phenomenon not addressed of current study in the type II pneumocytes. An alternative approach aiming to amplify and prolong the efficacy of the prostanoid–cAMP axis is the employment of phosphodiesterase inhibitors. Phosphodiesterases are known to limit the increase of cAMP levels by catalyzing the breakdown of the cyclic nucleotides in virtually all investigated cell types (22, 23). To test if rapid activation of cAMP-degrading enzymes is responsible for limiting the iloprost effect in ATII

cells, we analyzed the time course of cyclic nucleotide accumulation in the presence of the cAMP-specific PDE inhibitors rolipram and zardaverine. Indeed, a three- to fourfold increase in the iloprost-elicited cAMP response was noted, with levels of the cyclic nucleotide not declining within 60 min (the current observation period) after iloprost challenge. The response nearly approached that evoked by the most potent combination of forskolin and PDE inhibition. Cyclic nucleotide catabolism via phosphodiesterases is thus intimately involved in kinetics and extent of ATII responsiveness to prostanoid challenge. These data on the ATII prostanoid–cAMP axis translate well into the currently observed impact of iloprost and PGI2 on the characteristics of pneumocyte surfactant secretion. In the absence of stretching, even marked cAMP accumulation per se did not induce a lamellar body extrusion response. The physiological surfactant secretory response to cell surface distension was, however, markedly increased in the presence of the PGI prostanoids, and further amplified on coapplication of PDE III/IV inhibitors. The prostanoid-induced “amplification loop” was clearly related to the extent and duration of cAMP accumulation in the pneumocytes undergoing both physiological stretching and prostanoid exposure. Although the events underlying this synergism of biophysical and pharmacological challenge were not addressed in detail in the current investigation, cooperativity of stretch-induced calcium mobilization and possibly calcium–calmodulin-related activation of dependent protein kinases with cAMP-dependent phosphorylation of proteins involved in the secretory machinery, such as actin, is the most likely explanation, as hypothetically depicted in Figure 6. Like cAMP, the increase in intracellular cGMP levels in response to the NO donors SNAP and spermine NONOate was amplified in the presence of cGMP-specific PDE inhibitors. In

Rose, Zwick, Ghofrani, et al.: Stretch, Vasodilators, Surfactant Secretion in Pneumocytes

contrast to the prostanoid–cAMP axis, however, even pronounced elevation of the ATII cGMP content did not significantly affect surfactant secretion, either under baseline conditions or in response to the physiological stimulus of cell stretch, but rather a tendency toward decreased secretion was noted. These data do not exclude the possibility that long-term exposure of ATII cells to NO may alter the secretory response of this cell type, as currently only short-term studies were performed. The current finding is, however, well in line with the preceding observation that NO decreases both ATP content and surfactant synthesis in ATII cells within 2 h (18, 19). Exocytosis of surfactant contained in lamellar bodies is a critical event in the homeostasis of the alveolar surfactant system, suggested to be disturbed under conditions of inflammatory lung disease and pulmonary edema formation. Inhaled vasodilator therapy, targeting regional vasodilation in wellventilated lung areas, will at the same time affect type II pneumocytes located at the gas–liquid interface. The current study strongly suggests that, in this respect, prostanoid-based approaches substantially differ from NO-based approaches. Whereas the latter did not affect the surfactant secretory mechanisms in type II pneumocytes, even under circumstances of amplified cGMP accumulation due to PDE V inhibition, the prostanoid–cAMP axis impressively enhanced the surfactant secretion in response to the physiological stimulus of cell stretch. Maximum efficacy of this approach was noted on coadministration of the chemically stable PGI2 analog iloprost and type III/IV phosphodiesterase inhibition. Further studies are necessary to elucidate whether this prostanoid effect, adding to regional vasodilation, may be relevant and beneficial when employing inhalation of aerosolized prostanoids for selective pulmonary vasodilation in different lung diseases.

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